Cochlear outer hair cells enhance the sensitivity of mammalian hearing through active mechanical feedback powered by the membrane protein prestin. The long-term goal of our research efforts is to understand the mechanism by which prestin interacts with the membrane and responds to changes in the transmembrane potential. Preliminary research has established that prestin molecules self-associate, and that changes in prestin-prestin interactions alter the function and lateral organization of prestin in the membrane. We propose to extend these studies and further elucidate the relationship between the function of prestin and the formation of prestin complexes in the membrane. The first specific aim will be to determine the relationship between prestin oligomerization and function and establish if a particular oligomeric state represents the functional unit of prestin. To accomplish this aim we will utilize advanced optical imaging techniques, including fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP) and single molecule imaging. The function of prestin will be assayed by measuring the nonlinear capacitance. Native and mutated forms of prestin will be studied in order to delineate the molecular motifs that mediate prestin-prestin and prestin-membrane interactions. One focus of the mutational studies will be on regions in the C-terminal STAS domain that are predicted to participate in protein-protein interactions. In a second specific aim, we will determine whether prestin-prestin and prestin-membrane interactions depend on voltage. This will be accomplished by performing FRET, FRAP and FLIM experiments in voltage-clamped cells. The successful completion of these aims will represent the first direct determination of voltage-induced molecular changes in prestin organization and contribute a powerful new tool to prestin research. We will also investigate the effect of agents that perturb prestin function and alter membrane properties on prestin-prestin and prestin-membrane interactions, including non-steroidal anti-inflammatory agents (NSAIDs), which are known to induce hearing loss and tinnitus. The sensitivity of human hearing depends on the proper function of a motor protein called prestin located in sensory outer hair cells. The malfunction or absence of prestin, as may occur through altered prestin-prestin interactions by ototoxic compounds, results in high-frequency hearing loss. Because of the conservation of the part of the prestin molecule we will study, our research also has relevance for understanding the molecular basis of other diseases such as congenital diarrhea and skeletal abnormalities including dwarfism.